Antibodies and peptide antigens for producing antibodies having a selective binding specificity to bioactive intact parathyroid hormone (pth) 1-84

ABSTRACT

Peptide antigens corresponding to amino acid residues 2-12, 1-12, 2-15 and 1-15 of parathyroid hormone (PTH), antibodies having an affinity to such peptide antigens and methods of producing the same. Such antigens, antibodies and methods producing the same according to the present invention are useful in determining bioactive intact PTH levels in serum, plasma, and/or cell culture media. Such antibodies further possess a high degree of species cross-reactivity, but substantially mitigated cross-reactivity to non-whole PTH peptide fragments and little to no recognition of the first amino acid residue of PTH.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 12/286,620, filed Oct. 1, 2008 entitled ANTIBODIES AND PEPTIDEANTIGENS FOR PRODUCING ANTIBODIES HAVING A SELECTIVE BINDING SPECIFICITYTO BIOACTIVE INTACT PARATHYROID HORMONE (PTH) 1-84; which is acontinuation application of application Ser. No. 10/954,695, filed Sep.30, 2004, entitled ANTIBODIES AND PEPTIDE ANTIGENS FOR PRODUCINGANTIBODIES HAVING A SELECTIVE BINDING SPECIFICITY TO BIOACTIVE INTACTPARATHYROID HORMONE (PTH) 1-84; which is a continuation of U.S. Pat. No.6,838,264, issued on Jan. 4, 2005, entitled ANTIBODIES AND PEPTIDEANTIGENS FOR PRODUCING ANTIBODIES HAVING A SELECTIVE BINDING SPECIFICITYTO BIOACTIVE INTACT PARATHYROID HORMONE (PTH) 1-84, all of the teachingsof which are incorporated herein by reference.

STATEMENT RE: FEDERALLY SPONSORED RESEARCH/DEVELOPMENT

Not Applicable

BACKGROUND

Parathyroid hormone (PTH) and its importance in regulating theconcentration of calcium ions in the blood are well-known. In thisregard, such hormone is created by the parathyroid glands and, incombination with other factors, functions to regulate the blood calciumion levels such that the same is maintained in a steady concentration inboth cells and surrounding fluids. Essentially, PTH functions to releasestored calcium in the body when serum calcium levels decrease. On theother hand, such secretion is suppressed to the extent the serum calciumconcentration increases.

In its complete form PTH comprises a unique peptide comprised of 84amino acids. The specific sequence of PTH, as provided for a pluralityof species, namely, humans, rats, mice, bovids, dogs and pigs, aredepicted in FIG. 1, and a variation thereof in FIG. 2, which illustratesthe relatively consistent structure such hormone maintains between suchidentified species.

Given its significance in calcium metabolism for not only humans, but avariety of species, accurately measuring PTH has and continues to be ofsubstantial clinical significance. As is well-documented, serum PTHlevels serve as an important parameter for patients having diseases suchas hypercalcemia, primary hyperparathyroidism and osteoporosis, amongmany others. PTH likewise becomes of substantial clinical importance inpatients afflicted with chronic renal failure who, because of an excessin PTH production, can develop renal osteodystrophy.

Notwithstanding its important role in metabolism and clinicalsignificance, substantial difficulties have and continue to exist withregard to determinating circulating biologically active PTH levels.First of all, it is well-known that PTH is normally present at extremelylow levels, which are normally between 10 pg/ml to 65 pg/ml.Furthermore, it is known that the PTH peptide can be present in avariety of circulating PTH fragments, and in particular large non-(1-84)circulating PTH fragments which appear to co-migrate chromatographicallywith the (7-84) PTH molecule and are known to significantly interferewith conventional PTH assay measurements. Indeed, the large non-(1-84)PTH fragments may represent about one-half (2) of the PTH measured by amajority of current assays. Exemplary of the current shortcomings of theaccurate measurement of PTH are set forth in published PatentCooperation Treaty International Application No. PCT/US00/00855,International Publication No. W0/00/42437, entitled Methods forDifferentiating and Monitoring Parathyroid and Bone Status RelatedDiseases, and Lepage, Raymond et al., A Non-(1-84) CirculatingParathyroid Hormone (PTH) Fragment Interferes With Intact PTH CommercialAssay Measurement In Uremic Samples, Clinical Chemistry 44:4, 1998 pages805-809, the teachings of which are expressly incorporated herein byreference.

In an attempt to address such shortcomings, a new assay for detectingPTH levels was introduced by Scantibodies Laboratory, of Santee, Calif.,which incorporates a tracer antibody having a binding specificity forthe very end N-terminal fraction of human PTH, and more specifically,the first six amino acid residues thereof. As presently understood, suchassay appears to minimize cross-reactivity with large non-(1-84) PTHfragments. However, to derive such antibodies requires substantialeffort and expense in purifying the same. Moreover, such tracerantibodies have maximum recognition for only the first amino acidresidue of PTH, and substantially reduced specificity for any subsequentresidues thus obviating its use for some other species where the firstamino acid is different. Such drawbacks are discussed in the article byJohn, M. R. et al., entitled A Novel Immunoradiometric Assay DetectsFull-Length Human PTH but not Amino-Terminally Truncated Fragments:Implications for PTH Measurements in Renal Failure, The Journal ofClinical Endocrinology & Metabolism, Vol. 84, No. 11, 1999, p.4287-4290, the teachings of which are expressly incorporated herein byreference.

Thus, there has been and continues to be a long felt need in the art foran assay binding partner and method of generating the same that isspecific for bioactive intact PTH that can determine PTH levels withmitigated cross-reactivity to PTH peptide fragments. There is likewise aneed in the art for improved PTH binding partners that can measure PTHlevels in a more cost-effective manner and have a greater affinity forPTH that can be readily incorporated into immunoassay kits and the like.Still further there is a need for binding partners, namely, antibodieshaving a binding recognition that is specific to PTH and can be utilizedto detect PTH levels over a wide-variety of species. Finally, there is aneed in the art for an improved binding partner having a high bindingaffinity for PTH that may be readily derived using conventionalmechanisms that requires minimal purification, results in greaterbinding recognition for intact PTH, possesses minimal cross reactivityto large non-(1-84) PTH fragments, and can be derived utilizing methodsthat generate higher antibody yields than prior art binding partners.

BRIEF SUMMARY

The present invention specifically addresses and alleviates theabove-identified deficiencies in art. In this regard, the presentinvention is directed to certain novel antigens, antibodies, and methodsfor producing antibodies that are useful in determining bioactive intactPTH levels in a sample fluid, such as serum, plasma or cell culturemedia. The antigens, antibodies and methods of the present inventionhave the particular advantages of possessing greater affinity for PTH,and in particular, are designed to have a novel recognition for aminoacid residues extending beyond the first N-terminal PTH residue thanprior art antibodies specific to bioactive intact PTH, and further havenegligible cross-reactivity with the large non-(1-84) molecular forms ofPTH. Moreover, the antigens, antibodies and methods of producing thesame according to the present invention have substantialcross-reactivity with a wide variety of species, and may be utilized todetect PTH levels in not only humans, but also in rats, mice, bovids,dogs, and pigs.

According to a preferred embodiment, the antigen comprises the formulaVAL-SER-GLU-ILE-GLN-X-MET-HIS-ASN-LEU-GLY wherein X is selected from thegroup consisting of LEU [SEQ ID NO. 1] and PHE [SEQ ID NO. 2]. Withrespect to such embodiment, such antigenic peptide represents amino acidresidues 2-12 of PTH, with the sixth amino acid residue thereof beingselective between LEU and PHE, the former occurring in the PTH ofhumans, rats, mice and pigs, on one hand, and the latter, being inherentin the PTH of bovids and dogs, on the other hand. In a more highlypreferred embodiment, the antigen comprises a peptide having the formulaY-VAL-SER-GLU-ILE-GLN-X-MET-HIS-ASN-LEU-GLY wherein X is an amino acidresidue selected between LEU and PHE, as discussed above, and Y is anamino acid residue consisting of either SER or ALA [SEQ ID NO. 3, SEQ IDNO. 4, SEQ ID NO. 5, AND SEQ ID NO. 6, respectively], the formerreflecting the amino acid present in humans, dogs, and pigs, and thelatter being inherent in the PTH of rats, mice and bovines.

In further preferred embodiments, the antigen comprises the formulaVAL-SER-GLU-ILE-GLN-X-MET-HIS-ASN-LEU-GLY-LYS-HIS-LEU wherein X isselected from the group consisting of LEU [SEQ ID NO. 7] or PHE [SEQ IDNO. 8]. Such antigenic peptide represents amino acid residues 2-15 ofPTH, with the sixth amino acid residue comprising either LEU or PHE, tothus reflect the corresponding amino acid residue occurring in theappropriate species specified above. In another preferred embodiment,the antigenic peptide represents amino acid residues 1-15 of PTH andcomprises the formulaY-VAL-SER-GLU-ILE-GLN-X-MET-HIS-ASN-LEU-GLY-LYS-HIS-LEU, wherein Xcomprises amino acid residue LEU or PHE and Y is an amino acid residueconsisting of either SER or ALA [SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO.11, AND SEQ ID NO. 12, respectively], the latter being selective tocorrespond to a particular species identified above.

With respect to the antibodies and methods of producing the sameaccording to the present invention, such are directed to antibodieshaving an affinity and specificity for the aforementioned antigens.Preferably, the antibodies are specific to amino acid residues 2-12,1-12, 2-15 and 1-15, respectively, of PTH and are preferably producedvia the steps of administering a peptide antigen of the aforementionedvariety to a host animal, which preferably comprises a goat, to produceantibody production against the peptide antigen. In an alternative,preferred embodiment, antibody production is induced via theadministration of larger peptide fragments of PTH. Preferably, such PTHfragment may comprise (1-34) PTH, which may further optionally include acarrier protein covalently linked or fused thereto to increaseantigenicity or intact (1-84) PTH. To the extent antibodies are soughtto be derived to detect PTH in humans, the intact (1-84) PTH moleculepreferably comprises intact rat PTH or, to a lesser extent, human PTH.The antibody titer produced by the administration of the antigen to thehost animal is then monitored. Thereafter, antisera produced in the hostanimal is then isolated and selected such that the antibodies thereofhaving specificity for the desired antigenic region of PTH (i.e., aminoacid residues 2-12, 1-12, 2-15 and 1-15 of PTH, respectively) areisolated and thereafter purified. The antibodies may then be labeled orotherwise incorporated into any of a variety of conventional assays foruse in the detection of PTH, whether it would be for humans or any of avariety of species.

As will be recognized by those skilled in the art, the antigens,antibodies and methods of the present invention, by focusing on aminoacid residues 2-12, 1-12, 2-15 and 1-15 of PTH, respectively, focus onthat portion of the PTH molecule possessing the highest degree ofN-terminal biological activity, and thus maximize detection of the same.Moreover, with respect to the more highly preferred embodiments, byproviding antigens, antibodies and methods of producing the same thatare inclusive of other amino acid residues extending beyond theN-terminal biologically active site (i.e., up to and including thetwelfth (12th) and fifteenth (15th) amino acid residues of PTH), thespecificity and affinity of such antibodies are thus more highly refinedand enable the same to detect PTH levels with greater specificity thanprior art receptors, as incorporated into assays and the like. Perhapsmost importantly, however, the antibodies produced according to thepresent invention (as well as the methods and peptide antigens disclosedherein for achieving that end) have substantially minimizedcross-reactivity to large non (1-84) PTH peptide fragments, do notpossess maximum recognition for only the first amino acid residue ofPTH, and further, may be readily derived in a cost-effective mannerinsofar as the antibody yields generated from methods of the presentinvention should be greater than prior art methods.

It is therefore an object of the present invention to provide novelantigens for the production and isolation of antibodies, antibodies andmethods of producing antibodies that have a binding affinity andspecificity for PTH that possess mitigated cross-reactivity to large non(1-84) PTH peptide fragments.

Another object of the present invention is to provide novel antigens forthe production of antibodies, antibodies and methods of producingantibodies that have a greater affinity and specificity for PTH thanprior art binding partners and further, possess a higher degree of crossreactivity between inter-species PTH such that the antigens, antibodiesand methods producing the same according the present invention can bereadily utilized for the detection of PTH in a variety of species.

Another object of the present invention is to provide novel antigens forthe production and isolation of antibodies, antibodies and methods ofproducing antibodies which have a binding affinity and specificity formore of the biologically active N-terminal portion of PTH and, hence,are more effective and accurate in determining bioactive intact PTHlevels than prior art binding partners directed thereto.

Another object of the present invention is to provide novel antigens forthe production and isolation of antibodies, antibodies and methods ofproducing antibodies which do not possess a maximum binding affinity foronly the first N-terminal amino acid residue of PTH.

Another object of the present invention is to provide novel antigens forthe production and isolation of antibodies, antibodies and methods ofproducing antibodies that are less expensive to produce and generatehigher antibody yields than prior art methods for producing antibodieshaving a binding affinity and specificity for PTH.

Still further objects of the present invention are to provide novelantigens for the production of antibodies, antibodies and methods ofproducing antibodies which readily derive antibodies which may bereadily incorporated into any of a variety of commercially-availableassays and further, can be modified (e.g., to include a label, and thelike) as may be desired for any of a variety of immunoassayapplications.

BRIEF DESCRIPTION OF THE DRAWINGS

These, as well as other features of the present invention, will becomemore apparent upon reference to the drawings wherein:

FIG. 1 depicts the amino acid sequence of PTH for a variety of species,namely humans, rats, mice, bovids, dogs and pigs and further depictsamino acid sequences identified herein as SEQ ID NO. 1, SEQ ID NO. 2,SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO.7, SEQID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, and SEQ ID NO. 12.

FIG. 2 is an alternate illustration of FIG. 1 depicting a variation ofthe 1-84 amino acid sequence of PTH of the aforementioned species yetdepicting the conserved N-terminus of PTH wherein the amino acidsequences of SEQ ID NOS. 1-12 remain relatively constant.

FIG. 3 is a diagrammatical view of the N-terminal portion of human PTH.

FIG. 4 is a flow chart depicting the steps for producing antibodiesaccording to a preferred embodiment of the present invention.

DETAILED DESCRIPTION

The detailed description set forth below is intended as a description ofthe presently preferred embodiment of the invention, and is not intendedto represent the only form in which the present invention may beconstructed or utilized. The description sets forth the functions andsequences of steps for constructing and operating the invention. It isto be understood, however, that the same or equivalent functions andsequences may be accomplished by different embodiments and that they arealso intended to be encompassed within the scope of the invention.

The present invention encompasses antigens, antibodies and methods ofproducing antibodies that are directed to an antigenic region of PTHpositioned in the N-terminal region thereof, and more precisely, thefirst fifteen (15) amino acid residues extending from the N-terminaldepicted as 10 in FIGS. 1-3. As is well-known, the N-terminal region ofPTH is recognized as necessary for PTH/PTHrp receptor binding, and isfurther recognized as being most desirable epitope for measuringbioactive intact PTH levels, as may be found in a biological fluidsample, such as serum, plasma or cell culture media. Indicative of thecurrent state of the art associated with PTH and the methods ofdetecting the same are discussed at length in published PatentCooperation Treaty International Application No. PCT/US00/00855,International Publication No. W0/00/42437, entitled Methods forDifferentiating and Monitoring Parathyroid and Bone Status RelatedDiseases, and Lepage, Raymond et al., A Non-(1-84) CirculatingParathyroid Hormone (PTH) Fragment Interferes With Intact PTH CommercialAssay Measurement In Uremic Samples, Clinical Chemistry 44:4, 1998 pages805-809, the teachings of which are expressly incorporated herein byreference.

According to a preferred embodiment, the antigenic peptide of thepresent invention comprises those amino acid residues corresponding toamino acid residues 2-12 of PTH, collectively identified as 12 in FIGS.1-3. Specifically, such antigenic peptide will have the formula:

VAL-SER-GLU-ILE-GLN-X-MET-HIS-ASN-LEU-GLY

wherein X is an amino acid residue selected from either LEU [SEQ ID NO.1] or PHE [SEQ ID NO. 2]. As will be recognized by those skilled in theart, the sixth (6th) amino acid residue of this PTH peptide fragmentdoes differ between the cited species whereby such residue comprises LEUin humans, rats, mice and pigs, on one hand, but PHE for bovids and dogson the other. As will be appreciated by those skilled in the art,notwithstanding the single amino acid residue difference, such antigenicpeptide remains otherwise constant between the cited species which, asdiscussed more fully below, can enable antibodies to be prepared andultimately utilized that are cross-reactive and, hence, effective indetecting PTH levels in a variety of such species.

In a more highly preferred embodiment, the peptide antigen reflects thefirst twelve (12) amino acid residues of PTH, identified as 14, andcomprises the formula:

Y-VAL-SER-GLU-ILE-GLN-X-MET-HIS-ASN-LEU-GLY wherein X is an amino acidresidue selected from either LEU or PHE and Y is an amino acid residueselected from either SER or ALA [SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO.5, and SEQ ID NO. 6, respectively]. With respect to the variation at thefirst amino acid residue, it will be readily appreciated that suchantigenic peptide may be formed such that such amino acid comprises SER,as found in humans, dogs and pigs, or ALA, as found in rats, mice andbovids. In this respect, the variation provided for in the antigenicpeptide in the present invention, and in particular the more highlypreferred embodiments thereof, provide leeway such that the antibodiesultimately derived from such antigenic peptides may be formed to possessa higher binding affinity as may be desired to detect PTH in a givenspecies.

In more highly refined embodiments of the present invention, theantigenic peptides comprise sequences that correspond to amino acidresidue 2-15 and 1-15, respectively, of PTH. With regard to the former,identified in FIGS. 1-3 as 16, such antigenic peptide will have theformula: VAL-SER-GLU-ILE-GLN-X-MET-HIS-ASN-LEU-GLY-LYS-HIS-LEU wherein Xis an amino acid residue selected from either LEU [SEQ ID NO. 7] or PHE[SEQ ID NO. 8]. With respect to the latter embodiment corresponding toamino acid residues 1-15 of PTH, identified as 10, such antigenicpeptide will have the formula:Y-VAL-SER-GLU-ILE-GLN-X-MET-HIS-ASN-LEU-GLY-LYS-HIS-LEU wherein X is anamino acid residue selected from either LEU or PHE and Y is an aminoacid residue selected from either SER or ALA [SEQ ID NO. 9, SEQ ID NO.10, SEQ ID NO. 11, and SEQ ID NO. 12, respectively]. Notwithstanding theforegoing formulas for the aforementioned antigenic peptides, it will berecognized that the same will extend to all functional derivativesthereof, which is meant to include functionally comparable peptidesderived from the same region of PTH, as reflected in the sequences ofFIGS. 1-3, and having a similar ability to induce specific anti-PTHantibodies, and more particularly antibodies specific to the N-terminalamino acid residues of PTH. In this regard, such functional derivativemay be similarly positioned peptides or peptides derived from thesequences discussed above and reflected in FIGS. 1-3 havingsubstitutions, additions or deletions of amino acids, provided thederivation does not alter the ability of the peptide antigen to induceantibody reactive to PTH.

It should further be recognized that the peptide antigens of the presentinvention include those peptides whose amino acid sequence may beshifted within a few amino acids upstream or downstream of the antigenicpeptides discussed above in FIGS. 1-3, as well as those peptides havingconservative amino acid changes such that substitutions, additions ordeletion of amino acids or changes do not significantly effect theability of the peptide antigen to induce antibodies with high affinityand specificity for the first twelve amino acid residues of PTH, or anysub-components thereof.

With regard to antibody production, there is illustrated in FIG. 4 themethod 20 of generating the same. Initially, there is provided a peptide22 of the aforementioned variety which corresponds to all or a portionof the first twelve (12) to fifteen (15) amino acid residues of PTH of agiven species. According to a more highly preferred embodiment, however,the antigenic peptide utilized to generate antibody production willinclude larger PTH fragments, including but not limited to the (1-34)PTH and the entire intact (1-84) PTH peptide. Along these lines, whilethe antibodies ultimately produced will have specificity to those aminoacid residues corresponding to the aforementioned antigenic peptides(i.e., amino acid sequencing 2-12, 1-12, 2-15 and/or 1-15 of PTH) withinthe method of the present invention, at least with respect to theinitial step of providing an antigen 22 and subsequently administeringthe same, via step 24 discussed below, it will be appreciated that theentire PTH peptide and any fragment thereof can preferably serve as asuitable antigenic peptide to induce the production of antibodies whichwill ultimately possess the ideal binding affinity and specificity forthe biologically active portion of PTH at the N-terminal thereof. In amost highly preferred embodiment, the antigenic peptide provided andadministered will comprise the PTH fragment corresponding to amino acidresidues 1-34 of PTH, which may optionally be coupled to a carrierprotein. Such peptide fragment may alternatively preferably beadministered as whole length (1-84) PTH, which may take the form of anyof the species identified in FIGS. 1 and 2. Preferably, in the contextof developing antibodies specific for human PTH, the use of (1-34) ratPTH or whole length (1-84) rat PTH is preferred.

Such antigenic peptides may be produced by any of a variety of methodswell-known in the art, including synthesis by conventional methods, suchas solid-phase chemical synthesis or by recombinant technology. As willbe further appreciated, the synthetic peptides may optionally bechemically coupled to a carrier protein or, alternatively, recombinantpeptides may be generated as fusion proteins to increase antigenicity.As will be further appreciated by those skilled in the art, suchantigenic peptides may be screened based upon their ability to induceanti PTH antibody. In this respect, such screening techniques mayinclude, but not limited to, immunoprecipitation or immunoassay.

Once derived, such peptide antigens may then be utilized to generate theantibodies of the present invention using conventional techniques. Inthis regard, the peptide antigen(s), preferably in combination with anadjuvant, is/are administered to a host animal 24, which preferablycomprises a goat. It should be recognized, however, that other species,such as rabbits, mice, sheep, chicken, etc., may additionally beutilized as host animals. In this respect, the administration of suchantigens may be accomplished by any of a variety of methods, includingbut not limited to subcutaneous or intramuscular injection. As will beappreciated, the dose of peptide antigen administered willcorrespondingly vary with the specific peptide utilized, as well as theanimal host. As will additionally be recognized, however, in order toobtain antibodies possessing the highest affinity and specificitypossible for a given species, separate antibodies should be generatedagainst the appropriate comparable peptide from each species.

Once administered, the results of antibody titers produced in the hostanimal are then monitored 26, which may be conducted by any of a varietyof techniques well-known in the art, such as routine bleeds and thelike, with the antisera being isolated (e.g., via centrifugation), instep 28, and thereafter screened for the presence of anti-peptideantibodies having a binding affinity therefor. It will further berecognized that given the foregoing conventional immunological methodsfor deriving the antibodies of the present invention, such antibodiesmay be monoclonal or polyclonal in nature. Consistent with conventionalpractice it is preferred that the antisera be derived from a pluralityof host animals.

The resultant antisera derived from the host animal may be affinitypurified to derive the antibodies for the present invention. As iswell-known in the art, the antisera may be purified via conventionaltechniques, such as the introduction into a separation column with theaforementioned antigenic peptides corresponding to amino acid residuesequences 2-12, 1-12, 2-15 and/or 1-15 of PTH being bound to a solidphase (e.g., beads and the like). The antiserum may then be washed toremove antibodies not having specificity for the antigenic peptide orpeptides, with the remaining bound antibody, specific for the antigenicpeptide or peptides, ultimately being eluted therefrom. Such antibodymay then be stored per conventional practices well-known to thoseskilled in the art.

Advantageously, because the antibodies that are ultimately isolated willhave specificity for that antigenic region of PTH corresponding to aminoacid residue sequences 2-12, 1-12, 2-15 and 1-15, or any combinationthereof, the yield of antibodies generated via the methods of thepresent invention is believed to be substantially higher than antibodiesproduced via prior art methods whereby antibodies specific to smallerN-terminal sequences are generated. In this regard, due to the fact thatthe antibodies ultimately derived will be specific for amino acidresidues extending beyond the first six (6) to eight (8) amino acidresidues at the N-terminal of PTH provides a greater antigenic tertiarystructure for which such antibodies have developed an affinity, whetherit would be the entire 1-15 amino acid sequence or any portion thereof.

Moreover, to the extent such antibodies are isolated using thoseantigenic peptides corresponding to amino acid residue sequences 2-12and 2-15 of PTH, respectively, there will thus correspondingly beisolated antibodies that have not necessarily acquired a bindingaffinity, much less a maximum binding affinity, for the first amino acidresidue of PTH, which is known to be problematic of antibodies derivedvia prior art methods. Along these lines, the elimination or substantialsuppression of antibodies having an affinity for the first amino acidresidue of PTH will likewise be achieved by utilizing peptide sequencescorresponding to 1-12 and 1-15, respectively, of PTH provided that suchsequences are selected from those species having a dissimilar firstamino acid residue from the species for which the antibodies ultimatelyderive will be utilized. For example, in order to derive antibodiessuitable for detecting PTH levels in humans that lack in affinity forthe first amino acid residue of human PTH, it will be recognized thatthe antisera derived from the host animal may be purified againstpeptides corresponding to amino acid residue sequences 1-12 and 1-15 ofrat PTH. As will be understood, because the first N-terminal amino acidresidue of rat PTH comprises ALA, as opposed to SER found in humans, anyantibodies ultimately isolated will necessarily possess a bindingaffinity for the amino acid residues extending beyond such first aminoacid residue sequence.

Once derivatized in step 30, such antibodies may be used inimmunological techniques to correlate the presence of bioactive intactPTH as may be found in a given sample (e.g., serum or plasma). In thisrespect, the antibodies of the present invention may be used alone or incombination to screen a given sample to determine the presence ofbioactive intact PTH but yet advantageously avoid cross-reactivity withthe large non (1-84) PTH fragments. By way of example, such antibodiesmay be incorporated into an immunological assay kit. Exemplary of suchapplications include the human bioactive intact PTH and rat bioactiveintact PTH ELISA kits, produced by Immutopics, Inc., of San Clemente,Calif. which provide an enzyme-linked immunosorbent assay (ELISA) forthe quantitative determination of bioactive intact PTH levels in serum,plasma or cell culture media. In this regard, given the foregoingapplicability to derive antibodies specific to PTH for a variety ofspecies, it will be recognized that such immunological assay kits, suchas those provided by Immutopics, Inc., may be specifically designed suchthat the affinity and specificity of such antibodies apply to a widevariety of species or alternatively, generated against the appropriatecomparable peptide of a given species such that the kits are specificfor such species.

Additional modifications and improvements of the present invention mayalso be apparent to those of ordinary skill in the art. Thus, theparticular combination of parts described and illustrated herein isintended to represent only a certain embodiment of the presentinvention, and is not intended to serve as a limitation of alternativedevices within the spirit and scope of the invention.

1. A method for producing an isolated antibody having an affinity to theN-terminal portion of PTH (1-84), the method comprising the steps: a)administering rat (1-34) PTH peptide to a host animal to induce antibodyproduction against said rat (1-34) PTH peptide in said host animal,; b)monitoring antibody titer produced by said administration of said rat(1-34) PTH peptide to said host animal; c) extracting antisera producedin said host animal by said administration of said rat (1-34) PTHpeptide; and d) isolating and selecting said antibody from said antiseraextracted in step c) by affinity chromatography utilizing a peptideantigen selected from the group consisting of SEQ ID NO. 3 and SEQ IDNO.
 4. 2. The method of claim 1 further including the following step: e)attaching a detectable moiety to the antibody isolated and selected instep d).
 3. The method of claim 2 wherein said detectable moiety isselected from the group consisting of a radioactive tracer, afluorescent tracer, an enzymatic tracer and a dye tracer.
 4. The methodof claim 1 wherein the host animal is a goat.
 5. A method for producingan isolated antibody having an affinity to the N-terminal portion of PTH(1-84), the method comprising the steps: a) administering human (1-34)PTH peptide to a host animal to induce antibody production against saidrat (1-34) PTH peptide in said host animal; b) monitoring antibody titerproduced by said administration of said rat (1-34) PTH peptide to saidhost animal; c) extracting antisera produced in said host animal by saidadministration of said rat (1-34) PTH peptide; and d) isolating andselecting said antibody from said antisera extracted in step c) byaffinity chromatography utilizing a peptide antigen selected from thegroup consisting of SEQ ID NO. 5 and SEQ ID NO.
 6. 6. The method ofclaim 5 further including the following step: e) attaching a detectablemoiety to the antibody isolated and selected in step d).
 7. The methodof claim 6 wherein said detectable moiety is selected from the groupconsisting of a radioactive tracer, a fluorescent tracer, an enzymatictracer and a dye tracer.
 8. The method of claim 5 wherein the hostanimal is a goat.